Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum

Malaria remains a significant cause of human mortality with the vast majority of cases due to infection with Plasmodium falciparum [1]. Numerous efforts towards control and eradication of this disease are directed at different areas including insect vector control, vaccine development, and the discovery of new therapeutic drugs. All of these efforts benefit from a deeper understanding of the Plasmodium molecular biology.Transgene expression or allelic exchange of altered or tagged versions of genes as well as knockouts are essential tools to study gene function and genetic interactions. In yeast, for example, a whole genome knockout and GFP tagged collection quickly followed the sequencing of the genome and led to an explosive growth of functional analysis and protein localization studies in this organism [2-4]. In P. falciparum, such tools would provide a wealth of information and a valuable resource to the community but technical hurdles remain.Transient transfection of P. falciparum blood stages was reported 15 years ago [5] creating the possibility of transgene expression from an episomally maintained plasmid. Shortly thereafter stable transfection and homologous integration into the genome of drug-selectable constructs, was achieved [6]. Using genetically manipulated parasite lines, experiments including promoter analysis [7], localization studies [8], high-throughput drug screenings [9] and, importantly, the description of the first knockout [10] were possible, shedding light on important aspects of parasite biology, as well as its interaction with the human host and mosquito vector.Gene knockouts are especially important for demonstrating essentiality of putative drug targets. Without genetic validation of drug targets, substantial resources may be wasted in the pursuit of inhibitors for non-essential gene products. However, since the complete genome sequence of P. falciparum became available [11], no significant shift from single-gene towards systematic whol